Toll-like receptors (TLRs) regulate activation of the innate immune response and influence the formation of adaptive immunity by detecting and initiating signal transduction cascades in response to bacterial, viral, parasitic, and in some cases host-derived ligands (Lancaster et al., J. Physiol. 563:945-55, 2005). Members of the TLR family TLR1, TLR2, TLR4 and TLR6 are located on the plasma membrane and activate downstream signaling pathways in response to ligands including protein or lipid components of bacteria and fungi. TLR3, TLR7 and TLR9 are preferentially localized intracellularly, and respond to dsRNA, ssRNA and unmethylated CpG DNA, respectively.
TLRs signal through adaptor molecules myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain containing adaptor inducing interferon-beta (TRIF) and TRIF-related adaptor molecule (TRAM), initiating signaling pathways involving JNK/p38 kinase, interferon-regulatory factors (IFN) IFN-3, IFN-5 and IFN-7, and NF-kB, leading to the production of pro-inflammatory cytokines (Romagne, Drug Discov. Today 12:80-87, 2007). Dysregulation of TLR signaling is believed to cause a multitude of problems, and therapeutic strategies are in development towards this axis (Hoffman et al., Nat. Rev. Drug Discov. 4:879-880, 2005; Rezaei, Int. Immunopharmacol. 6:863-869, 2006; Wickelgren, Science 312:184-187, 2006). For example, antagonists of TLRs4, 7 and 9 are in clinical development for severe sepsis and lupus, (Kanzler et al. Nat. Med. 13:552-559, 2007).
TLR3 signaling is activated by dsRNA, mRNA or RNA released from necrotic cells upon inflammation or virus infection, and results in the induced secretion of interferons and pro-inflammatory cytokines, which have been associated with pathogen infections, and shown to contribute to a spectrum of inflammatory, immune-mediated and autoimmune diseases, for example colitis, asthma, psoriasis, septic shock, rheumatoid arthritis, inflammatory bowl disease and type I diabetes (Tabeta et al., Proc. Natl. Acad. Sci. 101:3516-3521, 2004; Underhill, Curr. Opin. Immunol. 16:483-487, 2004; Gaspari, J. Am. Acad. Dermatol. 54:S67-80, 2006; Van Amersfoort et al., Clin. Microbiol. Rev. 16:379-414, 2003; Miossec et al., Curr. Opin. Rheumatol. 16:218-222, 2004; Ogata and Hibi, Curr. Pharm. Res. 9:1107-1113, 2003; Takeda and Akira, J. Derm. Sci. 34:73-82, 2004; Doqusan et al. Diabetes 57:1236-1245, 2008). TLR3 expression has been shown to correlate with inflammatory responses associated with pathological conditions such as primary biliary cirrhosis of liver tissues (Takii et al., Lab Invest. 85:908-920, 2005). TLR3 also plays a key role in the immune responses upon virus infections; for example, TLR3 deficient animals display significantly reduced inflammatory mediators and a survival advantage over wild type animals upon influenza A virus infection (Le Goffic et al., PloS Pathog. 2:e53, 2006), and TLR3 deficient animals are protected from rotavirus infection-induced mucosal epithelial breakdown (Zhou et al. J. Immunology 178:4548-4556, 2007). In necrotic conditions, the release of intracellular content, including TLR3 ligand endogenous mRNA triggers inflammation expression of cytokines, chemokines and other factors to facilitate clearance of dead cell remnants and repair the damage. Necrosis often perpetuates chronic or aberrant inflammatory processes leading to secondary damage or cascade of effects.
Currently, a number of different approaches have been taken to target the activity of TLR3 for treatment of different indications. These approaches include TLR3 modulators such as agonists and antagonists, antibodies, peptides, TLR3 ligands dsRNA and poly(I:C), as well as functional analogs of these that target TLR3 activity. The potential indications for TLR3 antagonists include inflammatory conditions, sepsis, inflammatory bowel disease, inflammatory pulmonary disease, and autoimmune diseases. The potential indications and uses for TLR3 agonists include post-viral fatigue syndrome, glioma, prostate cancer, antiviral vaccines, bladder cancer, cervical dysplasia, human papilloma virus infection, breast cancer, viral infection prevention, tissue regeneration, and avian influenza vaccines.
Predictive pharmacokinetic, safety and efficacy studies will be required before any TLR3 modulator for human use can be brought to the market place. Such studies will involve both in vitro and in vivo testing in animal models of TLR3-associated pathologies. Lack of cross-reactivity of the modulators with TLR3s across species can pose a challenge in these studies. Thus, use of for example antibody-based TLR3 modulators may require evaluation of cross-reactivity of the antibodies between species, generation of surrogate antibodies against a TLR3 polypeptide expressed by a particular model animal, as well as significant in vitro characterization of such surrogate antibodies. Evaluation of cross-reactivity, surrogate generation and in vitro characterization will require the use of TLR3 polynucleotides and polypeptides from a suitable model animal. Importantly, the identification of suitable animal models for the above-mentioned studies requires the identification of animal species expressing TLR3 with high identity and homology to human TLR3.
Thus, a need exists for the identification of polynucleotides encoding TLR3 and TLR3 polypeptides being expressed in an animal model identified as suitable for the predictive pharmacokinetic, safety and efficacy studies of TLR3 modulators. A need also exists for related methods such as methods of expressing such polypeptides and testing the cross-reactivity of TLR3 modulators.